In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detection light emitted by the specimen as reflection or fluorescent light. The focus of an illumination light beam is moved in a specimen plane by means of a controllable beam deflector, normally by tilting two mirrors, whereby the deflection axes are usually at right angles with respect to each other so that one mirror deflects in the x direction while the other deflects in the y direction. The mirrors are tilted, for example, by means of galvanometer positioning elements. The output of the detection light coming from the object is measured as a function of the position of the scanning beam. Normally, the positioning elements are fitted with sensors in order to ascertain the actual position of the mirror. The illuminating light is coupled in by means of a beam splitter. The fluorescent or reflection light coming from the object passes through the beam splitter and subsequently reaches the detectors.
Especially in the case of confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope normally comprises a light source, a focusing lens system with which the light from the source is focused onto a pinhole diaphragm—the so-called excitation diaphragm—a beam splitter, a deflector for controlling the beam, a microscope lens system, a detection diaphragm and the detectors to pick up the detection or fluorescent light. The illuminating light is coupled in by means of a beam splitter. The fluorescent or reflection light coming from the object returns via the deflector to the beam splitter, passes through the latter and is subsequently focused on the detection diaphragm behind which the detectors are located. This detector arrangement is called a descanning arrangement. Any detection light that does not come directly from the region of focus takes a different light path and does not pass through the detection diaphragm, so that point information is obtained, which produces a three-dimensional image as a result of sequential scanning of the object with the focus of the illumination light beam. In most cases, a three-dimensional image is obtained by means of image data acquisition one layer at a time.
Leica's German Preliminary Published Application DE 199 06 757 A1 discloses an optical arrangement in the optical path of a light source suitable for fluorescence excitation, preferably in the optical path of a confocal laser scanning microscope, having at least one spectrally selective element for coupling the excitation light of at least one light source into the microscope and for blocking out the excitation light that is scattered and reflected off the object, or else the excitation wavelength coming from the object via the detection optical path. For purposes of attaining a variable configuration with a simple design, the arrangement is characterized in that excitation light having different wavelengths can be blocked out by the spectrally selective element. As an alternative, such an optical arrangement is characterized in that the spectrally selective element can be adjusted with respect to the excitation wavelength that is to be blocked out. Moreover, the cited publication states that the spectrally selective element can be configured as an AOTF (acousto-optical tunable filter) or as an AOD (acousto-optical deflector). The above-mentioned preliminary published application states that the spectrally selective element can cause a spatially spectral spreading out which can be compensated for, for instance, with three additional optical components.
German Preliminary Published Application DE 198 59 314 A1 discloses an arrangement of a light-diffraction element for separating excitation and emission light in the optical path of a microscope, preferably in a confocal microscope, and especially in a laser scanning microscope, whereby the light-diffraction element is traversed by the excitation light as well as by the emission light and influences at least one wavelength of the excitation by means of diffraction, whereas other wavelengths emitted by the specimen pass through the element without being affected, as a result of which they are spatially separated from the excitation light. This arrangement comprises an AOTF.
German Preliminary Published Application DE 199 44 355 A1 discloses an optical arrangement in the optical path of a laser scanning microscope with at least one spectrally selective element that can be adjusted to the wavelength of the excitation light of a light source, whereby said element couples excitation light of the light source into the microscope, blocks the excitation light that is scattered and reflected off an object out of the detection optical path and does not block out the detection light coming from the object. In order to simplify the design of the known arrangement as well as to expand the detection variants that are possible so far, this optical arrangement is characterized in that, downstream from the element, there is another optical component and, after it has been traversed, the dispersive and/or birefringent properties of the detection light are combined in such a way that they can be detected and, in a preferred embodiment, this is done coaxially.
In comparison to scanning microscopes where the illuminating light and the detection light are separated by means of a beam splitter, the scanning microscopes mentioned above entail the advantage of spectral flexibility since the acousto-optical component can be adjusted to any desired optical wavelength for the illumination or detection light by means of actuation with sound waves of different frequencies. In addition, the spectral separation of these microscopes is many times better than that of scanning microscopes with beam splitters.
A drawback of optical arrangements having an acousto-optical component for separating the illuminating light and the detection light as well as of scanning microscopes having an acousto-optical component for separating the illuminating light and the detection light lies in the fact that the acousto-optical component is birefringent, which leads to a detrimental splitting of the detection light beam. Moreover, the acousto-optical component usually has a prism effect, which causes a spectral splitting of the detection light beam. The known arrangements do not compensate for this effect adequately and they entail high losses of detection light output. Especially arrangements that call for three additional optical components for the compensation are expensive and their adjustment is complex.